Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation

Instituto de Biología Molecular y Celular de Plantas (CSIC.-UPV.), Ciudad Politécnica de la Innovación, Edf. 8E. C/. Ingeniero Fausto Elio s/n, Valencia, Spain.
Plant Cell Reports (Impact Factor: 3.07). 11/2009; 29(1):61-77. DOI: 10.1007/s00299-009-0798-8
Source: PubMed


Engineered male sterility in ornamental plants has many applications such as facilitate hybrid seed production, eliminate pollen allergens, reduce the need for deadheading to extend the flowering period, redirect resources from seeds to vegetative growth, increase flower longevity and prevent gene flow between genetically modified and related native plants. We have developed a reliable and efficient Agrobacterium-mediated protocol for the genetic transformation of different Kalanchoe blossfeldiana commercial cultivars. Transformation efficiency for cv. 'Hillary' was 55.3% whereas that of cv. 'Tenorio' reached 75.8%. Selection was carried out with the nptII gene and increasing the kanamycin concentration from 25 to 100 mg l(-1) allowed to reduced escapes from 50 to 60% to virtually 0%. This method was used to produce male-sterile plants through engineered anther ablation. In our approach, we tested a male sterility chimaeric gene construct (PsEND1::barnase) to evaluate its effectiveness and effect on phenotype. No significant differences were found in the growth patterns between the transgenic lines and the wild-type plants. No viable pollen grains were observed in the ablated anthers of any of the lines carrying the PsEND1::barnase construct, indicating that the male sterility was complete. In addition, seed set was completely abolished in all the transgenic plants obtained. Our engineered male-sterile approach could be used, alone or in combination with a female-sterility system, to reduce the invasive potential of new ornamentals, which has become an important environmental problem in many countries.

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    • "Mutation breeding did not play an important role in development of new commercial varieties (Descoings 2003). In recent years, several interesting traits have been introduced into K. blossfeldiana using genetic engineering resulting in production of compact and dwarf plants, plants with reduced ethylene-sensitivity, and male-sterile plants (Christensen et al. 2008; Garcia-Sogo et al. 2010; Lütken et al. 2010, 2011). "
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    ABSTRACT: Kalanchoë blossfeldiana and its cultivars are important worldwide as potted plants. Their improvement relies mainly on cross-breeding and interspecific hybridization, although the use of the resource of wild species was limited. The present study aimed to characterize novel features of interspecific hybrids that can increase attractiveness of these commercially important ornamental plants. We investigated the new traits of selected hybrids resulting from hybridization between K. blossfeldiana and three wild Kalanchoë species, namely K. nyikae, K. pubescens and K. marnieriana. The selected qualitative and quantitative traits of plant growth and flowering were analyzed in hybrids and parental species. The analysis demonstrated generally intermediate character of hybrids. Following principal component analysis, clear separation among plants forming three distinct clusters was observed, where hybrids were situated between the two parental species. The plants were clearly grouped according to flower characteristics, plant height and timing of flowering in all investigated groups. Quantitative analysis also revealed significant differences among parental plants and hybrids for all examined characteristics. Transgressive segregation and heterosis were observed in relation to specific traits such as days until first open flower, plant height, number of inflorescences, flower diameter and number of flowers. Pollen analysis of the interspecific hybrids revealed low viability of pollen or complete sterility. The interspecific hybrids exhibited novel features valuable for breeders. Thus, wide hybridization can contribute to the development of new cultivars and this study clearly demonstrates the usefulness of interspecific crosses in the improvement of Kalanchoë cultivars.
    Euphytica 04/2015; 205(3). DOI:10.1007/s10681-015-1441-0 · 1.39 Impact Factor
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    • "In all the lines of transgenic P. zonale plants expressing barnase under control of the PsEND1 promoter, we did not observe differences with respect to wild type plants in vegetative growth, flowering time or inflorescence number. Morphological analysis of the transgenic plants showed that, under greenhouse conditions, the expression of the PsEND1::barnase construct does not significantly affect the vegetative and floral development, thus confirming the anther specificity of the PsEND1 promoter region previously observed by means of the GUS expression studies in different dicots and monocots [38,39,41,42]. The potential biotechnological applications of the PsEND1 promoter largely depend on both its spatial and temporal expression pattern, since the ectopic expression of the cytotoxic agent would damage other plant tissues and organs, decreasing the agronomic value of hybrid plants. "
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    ABSTRACT: Background Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. Results The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3–4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. Conclusion The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.
    BMC Plant Biology 08/2012; 12(1):156. DOI:10.1186/1471-2229-12-156 · 3.81 Impact Factor
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    • "Therefore, a large number of anther-specific promoters and their key cis-elements from different plant species have been isolated and functionally analyzed, such as TA29 (Koltunow et al., 1990), NeIF-4A8 (Brander and Kuhlemeier, 1995), Zm13 (Hamilton et al., 1998), LAT52 (Muschietti et al., 1994), Osg6B (Yokoi et al., 1997), PyD3 (Xiao et al., 2006), PsEND1 (Roque et al., 2007) and MdAGP3 (Choi et al., 2010). Some of these anther-specific promoters, such as TA29, LAT 52, PsEND1 and key cis-element, for example, " anther-box " , have been already used for genetic engineering of male-sterile plants (Mariani et al., 1990; 1992; Van der Meer et al., 1992; Xiao et al., 2000; Roque et al., 2007; Garcı´a-Sogo et al., 2010). Anther7 gene of Arabidopsis theliana (ATA7) which putatively encodes a protein related to lipid transfer protein was found to express exclusively in flowers, and its mRNA was more pronounced in immature inflorescences than in mature flowers (Rubinelli et al., 1998). "
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    ABSTRACT: The 5'-flanking region of 1174 bp upstream of the translation start point (TSP) of a reported Arabidopsis anther-specific gene, Anther7 gene (ATA7), which putatively encodes a protein related to lipid transfer protein, was cloned and functionally analyzed in transgenic tobacco after been fused with -glucuronidase (GUS) gene reporter. Histochemical GUS staining of the transgenic plants showed that the cloned fragment did drive GUS expression exclusively in the anther, not in any other parts of floral organs, including pollens and nor in any vegetative tissue. Transverse section of the GUS-blue anthers disclosed that the blue cells were present uniquely in the tapetum of the anther. A series of 5'-deletion of cloned fragment indicated that a short segment of 179 bp upstream of the TSP (-155 bp upstream of the transcription start site) retained not only the promoter's driving power, but also its tapetum-specificity. Cis-acting element search in this short segment revealed the presence of numbers of organ-and tissue-specific motifs, including pollen-specific LAT52 and SLG13. These results indicated that the tapetum-specificity of ATA7 gene is mainly conferred by its promoter, and such a promoter, in particular, the core one should be useful both for identification of tapetum-involved genes and for biotechnological applications. Key words: Arabidopsis, Anther7 gene of Arabidopsis theliana (ATA7), anther-specific promoter, tapetum-specific promoter.
    AFRICAN JOURNAL OF BIOTECHNOLOGY 11/2010; 9(41):6826-6834. · 0.57 Impact Factor
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